Capillary zone electrophoresis (CZE) is an efficient analytical separation technique which utilizes differences in mobility of sample components in an electric field based on the electrical charge and molecular site and shape of the sample component. Conventional CZE systems typically comprise a buffer-filled capillary with outlet and inlet ends disposed in two reservoirs into which one sample is injected, a means for applying voltage to the capillary resulting in migration of the sample through the capillary, and a means for detecting the sample zone.
Sample injection systems and capillary zone electrophoresis channel systems have been integrated together on planar glass substrates for separation of sample components as described by Harrison et al. (1992, Anal. Chem. 64:19261932) and Seiler et al. (1993, Anal. Cem. 65:1481-1488). Additionally, capillary electrophoresis on microchips has been described by Manz et al., (1992, J. of Chromatography 593:253-258). Total chemical analysis systems (TAS) in which sample transport, chromatography or electrophoretic separations and detection are all performed have also been developed.
One of the limitations of conventional CZE is the extremely small amount of sample which must be used in order to obtain separation or resolution of sample components. The use of small volume samples results in low amount of sample components of interest representing a major limitation in the detectability of sample components. On the other hand, the larger the sample volume introduced into the capillary, the broader the sample component peaks will be. Attempts to increase injection sample volume typically leads to a breakdown in resolution due to broadening of the peaks attributable to individual sample components which one is actually trying to resolve or separate and possibly leads to generation of laminar flow inside the capillary.
A number of techniques have been developed for increasing the concentration of specific sample components of interest and narrowing the width of the injected sample. One such technique involves the use of a solid-phase adsorption medium followed by a sequential combination of pressure- and electrically-driven flows as described in U.S. Pat. No. 5,453,382. Using such a technique, the solution containing the sample component of interest is applied to the solid phase adsorption medium under conditions which permit sorption of the sample component of interest to the adsorption medium. The environment of the medium is then altered to promote desorption of the concentrated sample component and a voltage gradient is induced across the medium to induce electroosmosis. U.S. Pat. No. 5,340,452 also describes a similar method for increasing the concentration of sample components prior to electrophoresis by using an active material which selectively retains the sample components of interest at the inlet end of the capillary tube.
For some specialized samples, another obstacle to successful separation of components of a solution results from the low strength of the electric field in the buffer bordering the sample solution and the column buffer. To circumvent this problem, water or diluted buffer may be removed from the capillary or column using electro-osmotic flow while the sample components are stacked in a support buffer thereby concentrating the sample components in a sample with a minimum amount of laminar flow. Such a method is described in U.S. Pat. No. 5,116,471.
For a large volume samples in constrained containers, pressurized flow and countermigration can be used to increase the overall concentration as described by Hori et al. (1993, Anal. Chem. 65:2882-2286). The sample is introduced into a first vessel containing buffer which is connected to another vessel by a glass tube. An electrode extending into the first vessel applies a voltage to the sample while suction pressure is applied. The sample concentration increases throughout the first vessel rather than concentrating the sample in a discrete portion of that vessel because the applied potential field is unconstrained throughout the buffer volume. Because the concentration increase and electric fields are dispersed throughout the entire first vessel volume this technique is not applicable as a small volume injection/preconcentration technique. Moreover, this arrangement does not allow for micromanipulations such as electrophoretic separation within the vessel containing the concentrated sample.
Hence, none of the aforedescribed methods provide for concentration of sample components upon immediate introduction into a constrained small volume flow path which receives a fluid sample without the use of complicated systems such as discontinuous buffer systems and, in some instances, microengineered absorption devices. Accordingly, there exists a need in the art for more precise and efficient methods and devices for increasing the concentration of sample components of interest within a fluid sample while maintaining a consistent buffer and without microengineering absorption systems.